Interplay of endonucleolytic and exonucleolytic processing in the 3′-end formation of a mitochondrial nad2 RNA precursor in Arabidopsis

Abstract Initiation and termination of plant mitochondrial transcription are poorly controlled steps. Precursor transcripts are thus often longer than necessary, and 3′-end processing as well as control of RNA stability are essential to produce mature mRNAs in plant mitochondria. Plant mitochondrial 3′ ends are determined by 3′-to-5′ exonucleolytic trimming until the progression of mitochondrial exonucleases along transcripts is stopped by stable RNA structures or RNA binding proteins. In this analysis, we investigated the function of the endonucleolytic mitochondrial stability factor 1 (EMS1) pentatricopeptide repeat (PPR) protein and showed that it is essential for the production and the stabilization of the mature form of the nad2 exons 1–2 precursor transcript, whose 3′ end corresponds to the 5′ half of the nad2 trans-intron 2. The accumulation of an extended rather than a truncated form of this transcript in ems1 mutant plants suggests that the role of EMS1 in 3′ end formation is not strictly limited to blocking the passage of 3′-5′ exonucleolytic activity, but that 3′ end formation of the nad2 exons 1–2 transcript involves an EMS1-dependent endonucleolytic cleavage. This study demonstrates that the formation of the 3′ end of mitochondrial transcripts may involve an interplay of endonucleolytic and exonucleolytic processing mediated by PPR proteins.


INTRODUCTION
The mitochondria are the powerhouses of eukaryotic cells, and they contain a genome that is deri v ed from their bacterial ancestor. The expression of the few genes present in mitochondrial genomes, which number about 50 in most organisms, r equir es the activity of a complete gene expression system in the organelle. Gi v en the very limited coding capacity of mitochondrial genomes, this gene expression machinery requires importing a large number of nuclear-encoded mitochondria-targeted proteins. Many of these factors correspond to genes from the endosymbiont that have been transferred into the nucleus and whose protein products are transported from the cytosol into the organelle. Other mitochondrial factors are of nuclear-origin and have been selected throughout the evolution to assist mitochondrial gene expression. Mitochondrial gene expression thus involves a peculiar genetic organization consisting in expressing a highly degenerated bacterial scaffold (the mt genome) through the use of eukaryote-deri v ed functions. Such an intricate genetic organization involving two physically separate genetic compartments that obey to different evolutionary trends has shaped mitochondrial gene expression processes throughout evolution. In flowering plants, the fact that mitochondrial genomes mostly e volv e by genome r earrangements, r esulting in generally poorly conserved gene 5 and 3 untranslated regions (UTR) even at the within-species le v el, has played a role in the shaping of mitochondrial gene expression processes. Additionally, plant mitochondrial transcription appears to be a r elax ed process e xhibiting har dly any control or modulation both in its initiation and termination ( 1 , 2 ). Ther eby, pr ecursor mRNAs in plant mitochondria are often much longer than needed and posttranscriptional e v ents involving 5 and 3 processing as well as control of RNA stability are essential for proper gene expression in plant mitochondria. Several proteins belonging to the pentatricopeptide repeat (PPR) family have been shown to play roles in mature mRNA end formation in plant mitochondria (3)(4)(5). Unlike in plastids, where most genes are contained in long polycistronic transcripts ( 6 ) which r equir es numerous 5 and 3 RNA processing through both endonucleolytic and exonucleolytic cleavages to produce monocistronic mRNAs ( 7 ), mitochondrial transcripts in plants are most often produced as standalone transcripts. Lack of 5 -to-3 exoribonuclease activity in plant mitochondria causes 5 ends of mRNAs to correspond either to transcription start sites or to 5 -processed transcripts produced by endonucleolytic processing ( 4 , 8 ). Conversely, plant mitochondrial 3 ends are generated by 3 -to-5 exonucleolytic trimming of primary transcripts. In such a model, 3 -to-5 exonucleases like the polynucleotide phosphorylase (PNPase) digest mRNAs from their primary 3 ends until their progression is stopped either by a stable RNA secondary structure or a stabilizing protein like a PPR protein ( 9 ). Four of such PPR proteins called Mitochondrial Stability Factor 1, 2, 3 and 4 (MTSF1, 2, 3 and 4) were found to both set the 3 end and stabilize specific mature or precursor mitochondrial transcripts ( 5 , 10-12 ). In the present analysis, we characterized a new RNA stabilizing mitochondrial transfactor in Arabidopsis thaliana that we named Endonucleolytic Mitochondrial Stability factor 1 (EMS1) . We show that this PPR protein is essential for the stability of a nad2 precursor transcript and that unlike in other mutant stability factors longer and not shorter precursor transcripts accumulate in its absence, strongly suggesting that EMS1 recruits an endonuclease for 3 end processing of this nad2 pre-mRNA, thereby showing that endoribonucleolytic cleavage also plays a role in 3 end formation in plant mitochondria.

Primers
Oligonucleotides used in this study are listed in Supplementary Table S1.

Plant material and growth conditions
Arabidopsis ( Arabidopsis thaliana ) Col-0 plants were obtained from the Versailles Arabidopsis Stock Center. The N644391 ( ems1 ) Arabidopsis mutant line was acquired from the European Arabidopsis Stock Centre ( http://arabidopsis. info/ ). Homozygous ems1 mutants were genotyped by PCR using the primers listed in Supplementary Table S1 and the insertion site was confirmed by sequencing. Plants were grown on soil in a greenhouse under long-day conditions (16 h of light and 8 h of dark).

Functional complementation of ems1 mutants
For mutant complementation test, the full-length EMS1 coding sequence without its stop codon was amplified by PCR, cloned into the pDONR207 vector using the Gatewa y BP reaction (In vitro gen), and subsequentl y transferred into the pGWB5 binary vector ( 13 ). The resulting construct (35S::EMS1::GFP) was transformed into Agrobacterium tumefaciens C58C51 and introduced into heterozygous ems1 plants by the floral dip method ( 14 ). Functionally complemented homozygous mutant plants were identified among the obtained transgenic plants by PCR analysis.

Subcellular localization
The roots of functionally complemented homozygous mutant plants (see above for details) were used for subcellular localization analysis. Prior to observation, roots were soaked in 0.1 M Mitotracker ™ Red (Invitrogen) to label mitochondria. The GFP fluorescence was visualized in the transgenic cell lines by Leica TCS SP5 confocal microscopy with a 40 × 1.25 numerical aperture oil objecti v e. The filter set had an e xcitation wav elength / spectral detection bandwidth of 488 nm / 500 to 530 nm for GFP and 561 nm / 580 to 625 nm for Mitotracker ™ Red.

RN A e xtr action, r everse-tr anscription quantitative PCR and RNA gel blot
Total RNA was isolated from 8-week-old flower buds using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. RNA was treated with DNase Max (QIAGEN) w hen RN As wer e used in r e v erse transcription (RT)-PCR or quantitati v e R T-PCR (qR T-PCR) assays. Mitochondrial mRNA abundances wer e measur ed by qRT-PCR. They were calculated using the comparati v e Ct method after normalization to the nuclear 18S ribosomal RN A as previousl y described in ( 15 ). Two biological and three technical repeats were performed for these analyses.
For RNA gel blotting, 10 g of total RNA was electrophoretically separated in formaldehyde-containing (1.5% [w / v]) agarose gels and transferred onto nylon membranes (Genescreen) as described previously ( 10 ). Hybridization probes were generated by PCR amplification using gene-specific primers (listed in Supplementary Table S1) and radiolabeled using the Prime A Gene labeling kit (Promega) according to the manufacturer's recommendations.

Circular RT-PCR
Fi v e g of total RNA wer e cir cularized with 40 U of T4 RNA ligase (New England Biolabs), following the manufactur er's instructions. Cir cularized RNA was purified using the RNA Clean & Concentrator Kits (Zymo Research ®, California, USA). The first strand complementary DNA (cDNA) synthesis was done for 3 h at 40 • C using 400 U of M-MLV re v erse transcriptase (Fermentas), 8 mM of random hexamers (Eurofins), 1 × M-MLV buffer, 0.5 mM dNTPs and 40 U of Riboblock RNase inhibitor (Fermentas). The obtained cDNA was diluted four times, and 5 l Nucleic Acids Research, 2023, Vol. 51, No. 14 7621 of the obtained cDNA solution was used for PCR amplification with di v ergent primers. The primers used for mapping precursor transcripts are listed in Supplementary Table S1. Amplified PCR products were gel purified, cloned into pCR2.1 ® -TOPO ® TA vector (ThermoFisher Scientific) and inserts of independent recombinant plasmids were sequenced after E. coli transformation.

RNA immunoprecipitation assays
RN A imm unoprecipitation (RIP) experiments were performed using the MACS GFP-Tagged Protein Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions, with minor modifications. Succinctly, Arabidopsis cells expressing the 35S::EMS1::GFP translational fusion were collected from a 3-day-old culture and ground into a fine po w der in liquid nitrogen. Samples were homogenized in RIP lysis buffer (20 mM HEPES-KOH, pH 7.6, 100 mM KCl, 20 mM MgCl 2 , 1 mM DTT, 1% (v / v) Triton X-100, 1 × of complete EDTA-free protease inhibitor (Roche)) for 30 min at 4 • C with slow rotation (10 rpm). The lysates were clarified by ultracentrifugation at 100 000 g for 20 min at 4 • C, and 7 mg of proteins of the supernatant were incubated with 50 l of anti-GFP magnetic beads (Miltenyi Biotec) for 1 h at 4 • C with rotation (10 rpm). After four washes with 200 l RIP of washing buffer (lysis buffer containing 0.1% Triton X-100) and one wash with 100 l of washing buffer 2 from the kit, RNAs were extracted from the beads using TRI reagent (Life Technologies), precipitated with ethanol, and then used for RT-qPCR analysis. Prior to r etrotranscription, immunopr ecipitated RNAs wer e tr eated with DNase I and purified using the RNA Clean & Concentrator Kits (Zymo Research ® , California, USA). Two independent immunoprecipitations and three cDNA syntheses pr epar ed from each immunopr ecipitation were used in the analysis and a RIP experiment with untransformed PSB-D cells was used as a negati v e control.

Blue native gel and in-gel activity assays
Crude organelle extracts enriched in mitochondria were pr epar ed as previously described ( 11 ) using eight-week-old flower buds of ems1 mutant and Col-0 plants. One hundred micrograms of total proteins from purified organelles were loaded and separated on 4-16% (w / v) polyacrylamide Nati v ePAGE ™ Bis / Tris gels (Invitrogen). After electrophoresis, BN-PAGE gels were stained with Coomassie Blue or in buffers re v ealing the acti vities of mitochondrial respiratory complexes I and IV as previously described ( 16 ). Once sufficient coloration was obtained, gels were soaked in a fixing solution containing 30% (v / v) methanol and 10% (v / v) acetic acid to stop the reactions.

Immunoblot analysis
BN-PAGE gels were transferred to 0.45 m polyvinylidene difluoride (PVDF) membranes under liquid conditions in 50 mM Bis / Tris and 50 mM Tricine at 20 V overnight at 4 • C. Total proteins were extracted from the crude membrane in cold lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, of complete EDTA-free protease inhibitor (Roche)). Protein concentrations were determined with the Bradford method (Bio-Rad). A pproximatel y 30 g of total protein were resolved on SDS-PAGE and then transferred onto PVDF membrane under semi-dry conditions (Bio-Rad). Membranes were incubated with specific primary antibodies (Supplementary  Table S2) overnight a t 4 • C . Hybridiza tion signals wer e r evealed using enhanced chemiluminescence reagents (Western Lightning Plus ECL, Perkin Elmer).

The arabidopsis ems1 mutant displays a r etar ded growth phenotype
We originally identified the ems1 mutant from a collection of Arabidopsis mutants bearing T-DNA insertions in nuclear genes encoding mitochondrial-targeted PPR proteins. The affected PPR gene corresponds to AT3G09060 which encodes a PPR protein comprising 17 canonical Ptype repeats, according to the PlantPPR database ( http:// ppr.plantenergy.uwa.edu.au/ ). The T-DNA in the ems1 mutant (SALK 144391) is located in the gene segment encoding the tenth PPR r epeat, pr ecisely at position + 1926 relati v e to the start codon (ATG, A = +1) ( A genetic complementation test was then de v eloped to confirm that the observed mutant phenotype was due to the T-DNA insertion in the EMS1 gene. To this end, the full-length coding sequence of EMS1 was fused to the gr een fluor escent protein (GFP) tag under the control of the 35S promoter, yielding a 35S::EMS1::GFP construct that was then transformed into heterozygous ems1 plants. The resulting homozygous transgenic ems1 plants showed a r estor ed wild-type phenotype (Figure 1 B), supporting that the inactivation of the AT3G09060 gene is responsible for the defecti v e phenotype of the ems1 mutant.

The EMS1 protein is targeted to mitochondria in vivo
Based on the Arabidopsis subcellular database SUBA ( 17 ), the EMS1 protein harbors a putati v e mitochondrial targeting sequence. To verify its subcellular distribution, the in vivo localization of the EMS1::GFP translational fusion expressed in the ems1 complemented plants (Figure 1 B) was analyzed by confocal microscopy. The resulting GFP fluorescence signal was found to co-localize with the red signals of the MitoTracker (Figure 2 ), thus confirming the mitochondrial localization of the EMS1 protein in vivo .

Impaired complex I accumulation in ems1 mutants
The mitochondrial localization of the EMS1 protein encouraged us to consider that the de v elopmental alterations of ems1 mutants might result from impaired respiratory activity in these plants. The accumulation and activity of the differ ent r espiratory complex es wer e thus examined by b lue nati v e polyacrylamide gel electrophoresis (BN-PAGE). We observed a dramatic reduction in complex I accumulation in the ems1 mutant, both by in-gel activity staining and immunoblot analysis using an antibody against the carbonic anhydrase 2 (CA2), a complex I subunit (Figure 3 A). The observed reduction in complex I was restored in complemented plants, confirming that it was associa ted with inactiva tion of the EMS1 gene (Supplementary Figure S2). We did not detect any reduction in the accumulation of other r espiratory complex es (complex es III, IV and V), which in fact slightly overaccumulate in the mutant as compared to the wild type (Supplementary Figure S3).
The stead y-sta te le v els of se v er al subunits of respir atory complex es wer e also anal yzed by imm unoblot assay. As shown in Figure 3 B, two subunits of complex I (NADH DE-HYDROGENASE 7 and 9 (Nad7 and Nad9)) were reduced in the ems1 m utant, w hich is consistent with the decreased abundance and activity of complex I in these plants (Figure 3 A). Other proteins such as RISP (a subunit of complex III), CYTC (a mobile protein that shuttles between complex III and IV), Cox2 (a subunit of complex IV), and ATP-␤ (a subunit of complex V) accumulated at slightly higher or near normal le v els in mutant plants compared to the wild type. The r esults ar e consistent with the abundance of corresponding r espiratory complex es, as r e v ealed by BN-PAGE gel analysis (Supplementary Figure S3). The alternati v e respira tory pa thway is known to be induced when the mitochondrial electron transport chain is compromised. As expected, the stead y-sta te le v el of alternati v e oxidase (AOX) protein was found to be strongly increased in the ems1 mutant (Figure 3 B). Taken together, our results show that the loss of EMS1 activity affects the biogenesis of mitochondrial complex I, which in turn leads to the activation of the alternati v e respiratory pathway. EMS1 plays a role in the stability of the nad2 e x on 1-2 precursor transcript P-type PPR proteins are known to be involved in a wide range of RNA processing e v ents in plant organelles ( 3 , 18 ). The complex I deficiency in ems1 mutant lets us to hypothesize that EMS1 protein could have a role in the expression of one or se v eral mitochondria-encoded mRNAs encoding a complex I subunit. To this end, we first compared the stead y-sta te le v els of mature mitochondrial transcripts by RT-qPCR in ems1 mutant and Col-0 r efer ence plants.
Most mature mitochondrial mRNAs were found to accumula te a t the same or slightly higher le v els in ems1 compared to the wild type except for the RNA species spliced for nad2 intron 1 and intron 2 whose stead y-sta te le v els were gr eatly r educed in ems1 plants (Figur e 4 , RT-qPCR nad2 ex1-2 and nad2 ex2-3). The ma tura tion of the Arabidopsis nad2 mRNA results from the fusion of two distinct pre-mRNA molecules (called nad2 exons 1-2 and nad2 exon 3-5, see Figure 5 ) that ar e r eunified by a trans -splicing reaction implicating the two halves of nad2 intron 2, named 2a and 2b (Figure 5 A). To understand what causes the reduction in nad2 intron 1 and intron 2 splicing in ems1 plants, the steady le v el of these two precursor mRNAs was e valuated by RT-qPCR. This approach re v ealed opposite changes in the abundance of these two precursors, with an overaccumulation of nad2 exons 3-5 pre-mRNAs and an underaccumulation of na d 2 exons 1-2 precursors compared to the wild type (Figure 5 B). RNA gel blot analysis confirmed Com /Col-0) * *** *** * * * * Figure 4. nad2 transcripts spliced for introns 1 and 2 accumulate at reduced le v els in the ems1 mutant. The steady-state le v els of mature mitochondrial mRN As were anal yzed by RT-qPCR in wild-type (Col-0) and ems1 plants. The lo g 2 r atios of tr anscript accumulation in ems1 to wild-type ar e pr esented. For mRNAs without introns, a single RT-qPCR was performed, while for intron-containing transcripts, the accumulation of spliced transcript for each intron was examined. The analysis was based on three biological and three technical replicates per genotype, and standard errors are indicated. The data were normalized to the nuclear 18S rRNA gene. Significant differences are indicated with one ( P < 0.05) or three ( P < 0.001) asterisks. these observations (Figure 5 C). Such opposite behavior of the two precursor RNAs bracketing nad2 intron 2 did not support a role for EMS1 in the splicing of this intron. Effecti v ely, altered intron splicing is suspected when all involved precursor tr anscripts over accumulate (since they are less utilized in the splicing reaction) and the corresponding matur e mRNA decr eases in abundance accordingly. Instead, it suggested that the loss of mature nad2 mRNA was most likely due to instability of nad2 exons 1-2 precursors rather than a splicing defect of nad2 intron 2 itself. The overabundance of nad2 exons 3-5 precursor RNAs is consistent with this hypothesis since they cannot be processed in the transsplicing reaction involving nad2 intron 2 in the absence of the nad2 exons 1-2 precursors. Overall, these observations strongly suggested that the EMS1 protein plays most likely a key role in the stabilization of nad2 exons 1-2 precursor RNAs and not in the nad2 intron 2 splicing reaction per se .

The EMS1 protein binds within the 3 region of the nad2 e x ons 1-2 precursor transcript
To investigate a potential role of EMS1 in the stabilization of the nad2 exons 1-2 precursor, its RNA binding site was predicted using the previously established PPR recognition code ( 19 , 20 ). We applied the PPR recognition code to EMS1 repeats 2-17 and obtained a degenerate RNA recognition sequence covering all potential EMS1 binding sites (Figure 6 A). The obtained sequence was then used to scan the unedited and fully edited Arabidopsis mitochondrial genome. Interestingly, among the fiv e most likely identified binding sites, one (CCGCUGCUUACUGCUC) involved the nad2 transcript. This site was located in the first half of nad2 intron 2, while the other sites were mapped to non-coding regions (Figure 6 B). To determine whether EMS1 associates with this potential RNA target in vivo , RN A imm unoprecipitation (RIP) followed by RT-qPCR assa ys were perf ormed on extracts pr epar ed from the Ara- bidopsis cell line expressing the 35S::EMS1::GFP fusion, which proved to be functional (Figure 1 B). The EMS1-GFP fusion protein was immunoprecipitated with a GFP antibody and co-enriched RNAs were purified from the coimmunopr ecipitate. The r esulting RNAs wer e then used for cDNA synthesis and analyzed by qPCR using a set of primer pairs spanning nad2 intron 2a and 2b (Figure 6 C). Of all the regions tested, two showed highly significant enrichment, of which the one containing the predicted EMS1 binding sites was the most enriched (Figure 6 C). In addition, no co-IP enrichment was observed for any other mitochondrial introns, nor for any of the ribosomal RNAs or the nad9 mRN A w hose encoded protein stead y-sta te le v el was found to be reduced in ems1 plants (Supplementary Figure S4). These data provided strong support for the binding of EMS1 to the CCGCUGCUUACUGCUC sequence that is found 815 bases downstream of nad2 exon 2.

The 3 ends of nad2 e x ons 1-2 precursors coincide with the EMS1 binding site
The cause of the destabilization of the nad2 exons 1-2 precursor RNA in ems1 plants was further investigated by comparati v e mapping of its 3 extremity by circular RT-PCR in both ems1 and wild-type plants. Agarose gel analysis of the obtained amplification products re v ealed two bands of different size and intensity in Col-0 plants, w hereas onl y the larger one was detected in the ems1 mutant (Figure 7 A). Cloning and sequencing of the bands obtained in Col-0 plants showed that the small band corresponded to nad2 exons 1-2 transcripts terminating 835-837 nucleotides downstream of exon 2 (Figure 7 B) and that the larger band corresponded to longer nad2 exons 1-2 transcripts ending 894 nucleotides downstream of exon 2. Sequence analysis of the band obtained in ems1 showed that it corresponded to the longer form (+894) of the nad2 exons 1-2 transcripts (Figure 7 B). Quantification of the long transcripts by RT-qPCR showed that they are almost fiv e times more abundant in the ems1 mutant compared to wild-type (Figure 7 C). Taken together, our r esults r e v ealed that the nad2 exons 1-2 precursors carry 3 ends terminating either at position +835 / +837 or +894 downstream of exon 2. The production of the short transcripts is EMS1 dependent and their 3 ends coincide with the EMS1 RNA binding site.

The EMS1 protein is involved in the 3 -end formation of the 5 -half of nad2 intron 2
The r ecombinogenic natur e of plant mitochondrial genomes makes them prone to frequent sequence rearrangements, sometimes resulting in gene fragmentation when recombination occur within gene sequences ( 21 ). The most common type of gene splitting e v ent results from breaks within intron sequences, resulting in the production of independently transcribed gene fragments.
To reassemble a single, functional mature mRNA, the separ ate tr anscripts are joined back together by base-pairing interactions between the two intron halves, reforming a functional intron ( 22 , 23 ). Such trans -splicing reaction can only occur if the 5 and 3 half introns can re-associate into a splicing-competent structur e. Ther efor e, the 5 and 3 intron halves must be processed and trimmed to eliminate counterproducti v e 3 and 5 RNA e xtensions, respecti v ely. Our analysis of EMS1 strongly suggests that this protein plays a role in such processes by both processing and stabilizing the 3 end of pre-mRNAs carrying the 5 half of nad2 intron 2. We effecti v ely observ ed that ems1 plants are impaired in the production of nad2 transcripts spliced for intron 1 and 2, which are cis -and trans -introns, respecti v ely.
Howe v er, the lack of over-accumulation of the unspliced precursor carrying the 5 half of nad2 intron 2 was not in favor of a role of EMS1 in the splicing of this transcript but rather in its stabilization. A similar conclusion was drawn from the analysis of the Arabidopsis 3 -end processing PPR protein MTSF3, in which the destabilization of the nad2 exons 3-5 precursor in the corresponding mutant results, albeit indirectly, in the abolition of intron 2 splicing ( 5 ). This hypothesis was next supported by the binding site of EMS1 which coincides with the 3 extremity of the nad2 exons 1-2 transcript. By sitting on mRNA termini, PPR proteins were found to be able to act as physical barriers and thereby impede the progression of exonucleases along transcripts ( 9 ). In fact, plant mitochondrial transcription termination is known to show little or no modulation and can extend for several kilobases downstream of genes ( 1 ). Thus, unnecessary 3 sequence extensions r equir e exonucleolytic trimming via the action of 3 -to-5 exoribonucleases such as the PNPase or the RNase R homolog 1 to produce mature RNAs (24)(25)(26). In plant mitochondria, the type of transcripts stabilized by association with protecti v e PPR proteins at their 3 end includes mRNAs ( 5 , 10 , 12 ), but we have recently shown that precursor transcripts undergoing a trans -splicing reaction are similarly processed ( 11 ). EMS1 is indeed the second reported example of a pre-mRNApr otecting PPR pr otein, but its action in 3 end formation does not appear to only involve the blocking of 3 to 5 exonucleolytic trimming. In other previously reported cases the loss of the protecting PPR protein necessarily destabilizes the target RN A and, most lo gicall y, 3 -trimmed degradation RNA products are detected in the corresponding mutants ( 10 ). In the ems1 mutant, no 3 shorter but 3 longer nad2 exons 1-2 pr ecursors wer e detected (Figure 7 A and B). We found that these longer transcripts accumula te a t much lower le v els in wild-type than in ems1 mutant plants, suggesting that they have a certain degree of stability (Figure 7 C). The increased accumulation of elongated nad2 exons 1-2 pre-mRNAs in ems1 plants compared to the wild type suggests that EMS1 plays a role in their shortening, most likely by inducing endonucleolytic cleavage between positions + 835 and + 894, w hich m ust be followed by exonucleolytic trimming up to the EMS1 binding site. We also observed that longer transcripts (+894) accumulate to much lower le v els than + 835 precursors. The long transcripts thus appear to be inefficiently stabilized; enough nonetheless to be detected, but not to produce physiological le v els of nad2 e xons 1-2 precursors and thus Nad2 protein. Ther efor e, the 3 -end formation of nad2 exons 1-2 transcripts appears to involve both endonucleol ytic and exonucleol ytic processing e v ents, a mechanism of 3 -end formation not previously reported in plant mitochondria.

Analysis of the 3 end processing of transcripts carrying other 5 -half introns in arabidopsis
Fi v e trans -spliced introns including nad1 introns 1 and 3, nad2 intron 2, and nad5 introns 2 and 3 are found in Arabidopsis ( 23 ). To see if the definition of the 3 end of 5half introns follows the same molecular rules, the 3 termini of the corresponding precursor transcripts were mapped by circular RT-PCR. We also searched for the presence of short RNA footprints near the identified 3 termini in the clustered organellar sRN A (cosRN A) database ( 27 ), w hich may indicate stabilization of the corresponding transcripts by binding of PPR-like proteins to their ends. Effecti v ely, the binding of RNA stability factors, such as PPRs, has been shown to result in the accumulation of short RNA sequences corresponding to their binding sites in plant organelles ( 5 , 10 , 11 , 27-29 ). As shown previously, a cosRNA footprint can be found at the end of the nad1 exons 2-3 precursor, w hich perfectl y coincides with both the 3 end of the transcript and the binding site of the MTSF2 PPR, ensuring the stability of the molecule (Figure 8 A, ( 11 )). Our analysis supports a similar type of stability and processing mechanism for the precursor carrying the 5 half of nad5 intron 2, for which the identified 3 end overlaps with a cosRNA footprint (M97) (Figure 8 B). For the three other trans -intron precursors, the situation appears to be less straightforward, potentially implying different 3 end processing mechanisms. It first concerns the nad2 exons 1-2 precursor, for which no cosRNA matches its 3 end and the binding site of EMS1. The lack of cosRNA in this region may reflect a weaker binding of EMS1 to the transcript it protects compared to other stabilizing PPRs, such as MTSF2. A similar situation is found for the nad1 exon 1 precursor, stabilized by the MSP1 PPR protein ( 30 ). Although a cosRNA (M22) is found in the 5 half of nad1 intron 1, it is located 136 bases downstream of the 3 end of the precursor and thus the MSP1 binding site (Figure  8 C). We also found some variability regarding the 3 -end of the nad1 exon 1 precursor, suggesting imperfect transcript end protection by MSP1. The presence of a cosRNA (M22) downstream of MSP1 suggests, as for EMS1, that a twostep mechanism involving MSP1-mediated endoribonucleolytic cleavage followed by possible 3 to 5 trimming up to the MSP1 binding site may be responsible for the formation of the 3 end of the nad1 exon 1 pre-mRNA. The lack of cosRNA coinciding with both EMS1 and MSP1 binding sites may be indicati v e that they act similarly in 3 end RN A processing. Our anal ysis also re v eals an unclear 3end processing mechanism for the transcript carrying the 5 -half of nad5 intron 3. Effecti v ely, variab le 3 termini spanning over 14 bases were mapped at the end of nad5 exon 3 precursors and no cosRNA matching with these extremities could be identified (Figure 8 D), potentially excluding a protein-based stabilization mechanism for that transcript. The sequence conservation of 5 -half introns in angiosperms was next investigated in relation with the binding sites of PPR proteins involved in their stabilization. First, we found that the EMS1 protein and its RNA binding site are quite well conserved in angiosperms (Supplementary Figure S5). We also observed that the sequence of the 5 -half introns is globally conserved in angiosperms up to a region corresponding to the binding site of the factors responsible for their stability, although sequence insertions are sometimes found just downstream of the last exon in some species (Supplementary Figure S6). This break in DN A homolo gy passing the binding site of stabilizing PPR proteins suggests that these proteins are involved in protecting the integrity of 5 -half-intron sequences, since any recombination e v ent occurring upstream of their binding site would result in unstab le transcripts. Howe v er, the situa tion is dif ferent for the  Figure S6D). It thus appears that different series of e v ents account for the 3 -end processing and the stabilization of 5 -half intron precursors in plant mitochondria. The initial view was that the processing involves a protection by an RNA binding protein sitting on transcript's 3 end, blocking exonucleolytic degr adation. The conclusions dr awn from the analysis of the EMS1, which may thus also a ppl y to the precursor transcript protected by MSP1, indica te tha t a two-step processing mechanism comprising an endoribonucleolytic cleavage potentially followed by exonucleolytic trimming may also account for the 3 -end formation of pre-mRNAs in plant mitochondria.

DA T A A V AILABILITY
Sequence data from this article can be found in the GenBank / EMBL data libraries under accession numbers MTSF1, A T1G06710; MTSF2, A T1G52620; MTSF3, A T2G02150; MTSF4, A T4G19440; EMS1, A T3G09060; MSP1, AT4G20090.

SUPPLEMENT ARY DA T A
Supplementary Data are available at NAR Online.